Human Bcl-2 is a protein, which is closely associated with the process of programmed cell death (apoptosis). Apoptosis is an active, tightly regulated physiological process involved in development, normal cell turnover, and hormone-induced tissue athropy. Lack of programmed cell death plays an important role in cancer and other hyperproliferative diseases like restenosis, fibrosis, psoriasis or certain types of allergic diseases, in particular in tumour progression and, importantly, might contribute to the clinical problem of resistance to anti-neoplastic regimens, in particular standard chemotherapeutic compounds. In contrast to most normal tissues, in malignant tumours, such as a small cell lung cancer (SCLC) and non-small lung cancer (NSCLC), Bcl-2 is often co-expressed.
WO 95/08350 discloses anticode oligomers and methods of using them for controlling the growth of cancer cells expressing the Bcl-2 gene.
Klasa et al., Antisense & Nucleic Acid Drug Development 12: 1993-213 (2002) (review), discuss the biological effects of compound oblimersen sodium (G3139) and its potential as an antisense drug. The compound has the structure 5′-d(P-thio)TCT-CCC-AGC-GTG-CGC-CAT-3′ (SEQ ID NO: 65). Genta Incorporated submitted an NDA to the FDA for oblimersen sodium (G3139) plus dacarbazine (DTIC). It was based on an international, multi-center randomized, phase 3 study of oblimersen sodium (G3139) plus dacarbazine (DTIC) versus DTIC alone every three weeks as first-line chemotherapy for metastatic melanoma. In May 2004, it was reported that the study failed to show a survival benefit from the combination of G3139 plus DTIC. The combination arm was associated with increased toxicity and discontinuations due to adverse events (AEs) including 69 (18.6%) patients who discontinued therapy for adverse events on the G3139 arm versus 39 (10.8%) on the DTIC alone arm. The rate of serious adverse events, SAEs, was 40% on the G3139 arm versus 27% on DTIC alone. Since the dosing of DTIC was identical in the two arms; toxicity increases were likely due to the addition of G3139. Survival was not improved and toxicity was increased. The NDA was subsequently withdrawn. However, the sponsor's analysis of secondary endpoints did show a statistically significant benefit in progression-free survival from a median of 49 days on DTIC to 74 days on the combination, a difference of 25 days (p=0.0003, HR=0.73). Also, the sponsor reported a significant difference in response rate of 6.8% for DTIC alone versus 11.7% for the combination (p=0.019). The fact that oblimersen sodium fulfilled the secondary endpoint indicates that it could have been an effective compound for the treatment of metastatic melanoma. The increased toxicity, the selection of primary endpoint and the overall clinical trail design were all factors that contributed to the failure.
LNA containing oligonucleotides targeting the 6 first codons of the human Bcl-2 mRNA were studied in a Ph.D. thesis defended by Jan Stenvang Jepsen (May 2003, University of Copenhagen). Fully modified LNA phosphodiester (PO) sequences, phosphorodiester headmers (LNA/PO at the 5′-end and DNA/PS phosphorothioate at the 3′-end), fully phosphorodiester gapmers (gap sizes of 8, 10, 12, 14) and gapmers with exclusive thiolation in the gap (gap sizes of 8, 10, 12, 14) were assayed for in vitro uptake with different transfecting agents and for down-regulation of Bcl-2 protein. The uptake study was performed in MCF-7 cells and the results were analyzed by microscopy and flow-cytometry. Equally efficient delivery was obtained for all the different PO and PO/PS containing constructs. Although a variety of LNA-containing oligonucleotides and constructs were studied, Stenvang Jepsen did not disclose or anticipate LNA-containing oligonucleotide gapmers wherein a substantial number of the nucleotides links in the target binding domain, including the LNA flanks, were phosphorothioate groups (—O—P(O,S)—O—), probably because it was known that phosphorothiolation would cause a reduction of affinity and because no stability problems were identified.
Frieden et al., Nucleic Acid Research, 2003, Vol. 31, No. 21, 6365-6373, and WO 2004/046160 A2 disclose various considerations with respect to the design of antisense oligonucleotides based on in vitro experiments.
Fluiter et al., Nucleic Acid Research, 2003, Vol. 3, 953-962, discloses in vivo tumour growth inhibition and biodistribution studies of LNA antisense oligonucleotides.